Method for opening tight junctions

ABSTRACT

The use of antagonists to JAM-1 Claudin-4 and occludin to open tight junctions. The antagonists include, by way of example antibodies and antibody fragments that bind to the proteins and small interfering nucleic acids that downregulate the mRNA encoding the proteins.

This claims the benefit under 35 U.S.C. § 119 (e) of U.S. ProvisionalPatent Application Ser. No. 60/529,682 filed on Dec. 15, 2003 the entirecontents of which is incorporated by reference.

BACKGROUND OF THE INVENTION

The teachings of all of the references cited herein are incorporated intheir entirety by reference.

A major disadvantage of drug administration by injection is that trainedpersonnel are often required to administer the drug. Forself-administered drugs, many patients are reluctant or unable to givethemselves injections on a regular basis. Injection is also associatedwith increased risks of infection. Other disadvantages of drug injectioninclude variability of delivery results between individuals, as well asunpredictable intensity and duration of drug action.

Despite these noted disadvantages, injection remains the only approveddelivery mode for a many important therapeutic compounds. These includeconventional drugs, as well as a rapidly expanding list of peptide andprotein biotherapeutics. Delivery of these compounds via alternateroutes of administration, for example, oral, nasal and other mucosalroutes, often yields variable results and adverse side effects, andfails to provide suitable bio-availability. For macromolecular speciesin particular, especially peptide and protein therapeutics, alternateroutes of administration are limited by susceptibility to inactivationand poor absorption across mucosal barriers.

Mucosal administration of therapeutic compounds may offer certainadvantages over injection and other modes of administration, for examplein terms of convenience and speed of delivery, as well as by reducing orelimination compliance problems and side effects that attend delivery byinjection. However, mucosal delivery of biologically active agents islimited by mucosal barrier functions and other factors. For thesereasons, mucosal drug administration typically requires larger amountsof drug than administration by injection. Other therapeutic compounds,including large molecule drugs, peptides and proteins, are oftenrefractory to mucosal delivery.

The ability of drugs to permeate mucosal surfaces, unassisted bydelivery-enhancing agents, appears to be related to a number of factors,including molecular size, lipid solubility, and ionization. Smallmolecules, less than about 300-1,000 Daltons, are often capable ofpenetrating mucosal barriers, however, as molecular size increases,permeability decreases rapidly. Lipid-soluble compounds are generallymore permeable through mucosal surfaces than are non-lipid-solublemolecules. Peptides and proteins are poorly lipid soluble, and henceexhibit poor absorption characteristics across mucosal surfaces.

In addition to their poor intrinsic permeability, large macromoleculardrugs, including proteins and peptides, are often subject to limiteddiffusion, as well as lumenal and cellular enzymatic degradation andrapid clearance at mucosal sites. These mucosal sites generally serve asa first line of host defense against pathogens and other adverseenvironmental agents that come into contact with the mucosal surface.Mucosal tissues provide a substantial barrier to the free diffusion ofmacromolecules, while enzymatic activities present in mucosal secretionscan severely limit the bioavailability of therapeutic agents,particularly peptides and proteins. At certain mucosal sites, such asthe nasal mucosa, the typical residence time of proteins and othermacromolecular species delivered is limited, e.g., to about 15-30minutes or less, due to rapid mucociliary clearance.

In summary, previous attempts to successfully deliver therapeuticcompounds, including small molecule drugs and protein therapeutics, viamucosal routes have suffered from a number of important and confoundingdeficiencies. These deficiencies point to a long-standing unmet need inthe art for pharmaceutical formulations and methods of administeringtherapeutic compounds that are stable and well tolerated and thatprovide enhanced mucosal delivery, including to targeted tissues andphysiological compartments such as central nervous system. Morespecifically, there is a need in the art for safe and reliable methodsand compositions for mucosal delivery of therapeutic compounds fortreatment of diseases and other adverse conditions in mammaliansubjects. A related need exists for methods and compositions that willprovide efficient delivery of macromolecular drugs via one or moremucosal routes in therapeutic amounts, which are fast acting, easilyadministered and have limited adverse side effects such as mucosalirritation or tissue damage.

In relation to these needs, an especially challenging need persists inthe art for methods and compositions to enhance mucosal delivery ofbiotherapeutic compounds that will overcome mucosal epithelial barriermechanisms. Selective permeability of mucosal epithelia has heretoforepresented major obstacles to mucosal delivery of therapeuticmacromolecules, including biologically active peptides and proteins.Accordingly, there remains a substantial unmet need in the art for newmethods and tools to facilitate mucosal delivery of biotherapeuticcompounds. In particular, there is a compelling need in the art for newmethods and formulations to facilitate mucosal delivery ofbiotherapeutic compounds that have heretofore proven refractory todelivery across mucosal barriers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Screening siRNAs against TJ proteins identifies those thataffect TER. Three siRNAs against each TJ protein expressed inrespiratory cells were designed and chemically synthesized. The effectson TER were monitored before and after transfections. The figure is anexample of the screening of siRNAs against CLDN 12 and JAM-1. The siRNAagainst CLDN 4 was used as a positive control. The best candidatesshowing maximum effect were used for determining the extent of mRNAreduction by the bDNA method.

FIG. 2. Time Course of Specific siRNAs and Lipofectamine alone (blank)on TER. (A) The percent of TER, normalized against control siRNA at eachtime point. Effects of the transfection reagent (lipofectamine) on TERcan be corrected. (B) Although the negative effects on TER bytransfection procedure and reagents (blank) are observed, the TER valuereturns to normal after 3-4 days of transfection.

FIG. 3. Transfection of Selected siRNAs Against TJ Targets DecreasesTER. 16HBE14 cells were grown on 12-well inserts for 3 days prior totransfection (80 pmol/insert). TER values measured at day 4 aftertransfection were near the optimal time point of TER recovery aftertransfection.

FIG. 4. CLDN 4 Knockdown Decreases TER. (A) Epithelial cells were grownon 12-well inserts to the stage where tight junctions formed asindicated by values for transepithelial-electrical resistance (TER).siRNAs against claudins 4 and 12 and a negative control (80 pmol/each)were used individually to transfect cell monolayers. (B) Epithelialcells were grown in 6-well plates to 50% confluence followed bytransfections with siRNA against claudin 4 and a negative control. TERvalues before day 9 are not shown since TER does not significantlydevelop until a mono-layer is established and tight junctions areformed.

FIG. 5. Knockdown of Either CLDN 1 or 12 with CLDN 4 Decreases TER.Results indicate that ½ dose (40 pmol) did not show any significanteffects on TER for any siRNA alone, while higher dose (80 pmol) of siRNAagainst CLDN 4 yields a knockdown effect. The effects of equal amountsof CLDN 4 and CLDN 1 (40 pmol each) is similar to 80-pmol of siRNAagainst CLDN 4 alone, while siRNA combinations with CLDN 12 (generallyineffective at any dose) have little effect.

FIG. 6. Knockdown of Either CLDN 4 or 12 with JAM-1 Decreases TER.Results indicate that ½ dose (40 pmol) did not show any significanteffects on TER for any siRNA alone, while higher dose (80 pmol) siRNAagainst CLDN 4 yields a knocking down effect. The effects of equalamount of CLDN 4 and JAM 1(40 pmol each) is similar with 80-pmol ofsiRNA against CLDN 4 alone, while pairing with siRNA against CLDN 12(generally ineffective at any dose) has little effect.

FIG. 7. Increased Dextran Permeability in siRNA Transfected CellsCorrelates with Decreases in TER. Permeability assays were conducted onday 5 after transfection of 16HBE14o- Cell Inserts with siRNAs againstindicated targets, using FITC-dextran, MW 4,400 in MEM/f12 media. Thechanges in permeability correlated well with changes in TER values (FIG.6).

FIG. 8. Knockdown of Either CLDN 4 or 12 and Occludin Decreases TER.Results indicate that ½ dose (40 pmol) did not show any significanteffects on TER for any siRNA alone (see FIGS. 5 and 6), while higherdose (80 pmol) siRNA against CLDN 4 yields a knockdown effect. Theeffects of equal amounts of CLDN 4 and occludin (40 pmol each) issimilar to 80 pmol of siRNA against CLDN 4 alone, while combination withsiRNA against CLDN 12 (generally ineffective at any dose) has littleeffect.

DESCRIPTION OF THE INVENTION

The instant invention satisfies the foregoing needs and fulfillsadditional objects and advantages by providing novel pharmaceuticalcompositions methods that open the tight-junctions in the nose. Thesecompositions are antagonists to either the extracellular domain of theJAM-1protein or the extracellular domain of the claudin-4 protein or theextracellular domain of the occludin protein. Examples of these areantibodies, antibody fragments and single chain antibodies that bind toeither junctional adhesion molecule- I (JAM-1), occludin or claudin-4.The permeabilizing agent reversibly enhances mucosal epithelialparacellular transport, typically by modulating epithelial junctionalstructure and/or physiology at a mucosal epithelial surface in thesubject. This effect typically involves inhibition by the permeabilizingagent of homotypic or heterotypic binding between epithelial membraneadhesive proteins of neighboring epithelial cells. Also, smallinterfering nucleic acids can be used to downregulate the peptidesthrough the mechanism of RNA interference as described below and thusopen the tight junctions.

Epithelial cells provide a crucial interface between the externalenvironment and mucosal and submucosal tissues and extracellularcompartments. One of the most important functions of mucosal epithelialcells is to determine and regulate mucosal permeability. In thiscontext, epithelial cells create selective permeability barriers betweendifferent physiological compartments. Selective permeability is theresult of regulated transport of molecules through the cytoplasm (thetranscellular pathway) and the regulated permeability of the spacesbetween the cells (the paracellular pathway).

Intercellular junctions between epithelial cells are known to beinvolved in both the maintenance and regulation of the epithelialbarrier function, and cell-cell adhesion. The tight junction (TJ) ofepithelial and endothelial cells is a particularly important cell-celljunction that regulates permeability of the paracellular pathway, andalso divides the cell surface into apical and basolateral compartments.Tight junctions form continuous circumferential intercellular contactsbetween epithelial cells and create a regulated barrier to theparacellular movement of water, solutes, and immune cells. They alsoprovide a second type of barrier that contributes to cell polarity bylimiting exchange of membrane lipids between the apical and basolateralmembrane domains.

Tight junctions are thought to be directly involved in barrier and fencefunctions of epithelial cells by creating an intercellular seal togenerate a primary barrier against the diffusion of solutes through theparacellular pathway, and by acting as a boundary between the apical andbasolateral plasma membrane domains to create and maintain cellpolarity, respectively. Tight junctions are also implicated in thetransmigration of leukocytes to reach inflammatory sites. In response tochemo-attractants, leukocytes emigrate from the blood by crossing theendothelium and, in the case of mucosal infections, cross the inflamedepithelium. Transmigration occurs primarily along the paracellular routand appears to be regulated via opening and closing of tight junctionsin a highly coordinated and reversible manner.

Numerous proteins have been identified in association with TJs,including both integral and peripheral plasma membrane proteins. Currentunderstanding of the complex structure and interactive functions ofthese proteins remains limited. Among the many proteins associated withepithelial junctions, several categories of trans-epithelial membraneproteins have been identified that may function in the physiologicalregulation of epithelial junctions. These include a number of“junctional adhesion molecules” (JAMs) and other TJ-associated moleculesdesignated as occludins, claudins, and zonulin.

JAMs, occludin, and claudin extend into the paracellular space, andthese proteins in particular have been contemplated as candidates forcreating an epithelial barrier between adjacent epithelial cells andchannels through epithelial cell layers. In one model, occludin,claudin, and JAM have been proposed to interact as homophilic bindingpartners to create a regulated barrier to paracellular movement ofwater, solutes, and immune cells between epithelial cells.

A cDNA encoding murine junctional adhesion molecule-1 (JAM-1) has beencloned and corresponds to a predicted type I transmembrane protein(comprising a single transmembrane domain) with a molecular weight ofapproximately 32-kD, Williams, et al., Molecular Immunology 36:1175-1188 (1999); Gupta, et al., IUBMB Life, 50: 51-56 (2000); Ozaki, etal., J. Immunol. 163: 553-557 (1999); Martin-Padura, et al., J Cell Biol142: 117-127 (1998). The extracellular segment of the molecule comprisestwo Ig-like domains described as an amino terminal “VH-type” and acarboxy-terminal “C2-type” carboxy-terminal β-sandwich fold, Bazzoni etal., Microcirculation 8:143-152 (2001). Murine JAM-1 also contains twosites for N-glycosylation, and a cytoplasmic domain. The JAM-1 proteinis a member of the immunoglobulin (Ig) superfamily and localizes totight junctions of both epithelial and endothelial cells.Ultrastructural studies indicate that JAM-1 is limited to the membraneregions containing fibrils of occludin and claudin.

Transfection of a murine JAM-1-encoding cDNA into CHO cells leads tolocalization of the JAM-1 protein at cell-cell contacts, which onlyoccurs in confluent monolayers when neighboring cells express JAM. Inmixed cultures, where JAM transfectants are in contact with controltransfectants, the protein remains diffuse—suggesting that JAMclustering is due to homophilic interaction, Martin-Padura, et al., JCell Biol 142: 117-127 (1998).

Experimental evidence suggests that JAM-1 can mediate homotypic adhesionand influence monocyte transmigration via heterotypic adhesive andde-adhesive interactions. A monoclonal antibody against murine JAM-1inhibits transmigration of leukocytes across endothelial cells and in anin vivo model of skin inflammatory reaction, Martin-Padura, et al., JCell Biol 142: 117-127 (1998). Anti-murine JAM-1 antibodies also inhibitaccumulation of leukocytes in the cerebrospinal fluid incytokine-induced meningitis. It is unknown how these effects aremediated. In one model, the antibodies may inhibit a heterotypicinteraction between JAM-1 and a leukocyte receptor, Del Maschio et al.,J. Exp. Med. 190:1351-1356 (1999). Alternatively, the anti-JAM-1antibodies may stabilize a homophilic JAM-mediated interaction betweenneighboring cells and thereby inhibit dissociation of the junctionalcomplex, Balda et al., Cell Devel. Biol. 11: 281-289 (2000).

One model for JAM-1 activity proposes that an extracellular domain ofJAM-1 is involved in intercellular adhesive interactions. Formation ofJAM-1 dimers is thought to be due to stable and noncovalentinteractions. Dissociation of JAM-1 dimers into monomeric subunits isreported at high ionic strength and acidic pH. In this general model,JAM-1 dimers are hypothesized to act as building blocks forJAM-1-dependent homophilic adhesion. In particular, JAM-1 may dimerizein cis-interactions yielding parallel homodimers positioned at one cellsurface, and the cis-dimerization might expose an interface availablefor homophilic adhesive interactions between JAM-1 molecules on opposingcell surfaces. This model could account for homotypic adhesion betweenadjoining cells within confluent endothelial or epithelial monolayers.In addition, JAM-1 dimers expressed on transmigrating leukocytes areproposed to interact with JAM-1 dimers expressed on endothelial cells,thus accounting for the adhesion and de-adhesion events that occurduring leukocyte transendothelial migration, Dejana, et al., Throb.Haemost. 86: 308-315 (2001).

The putative extracellular domain of human JAM-1 was recently expressedas a fusion protein to generate anti-human JAM-1 antibodies thatinhibited transepithelial resistance recovery (TER) in T84 cellmonolayers after tight junction disruption mediated by transient calciumdepletion, Liu et al., J. Cell. Sci. 113:2363-2374 (2000). Inparticular, the anti-JAM antibodies inhibit JAM-1 and occludinredistribution to TJs following calcium mediated disruption. However,these authors report that purified recombinant human JAM-1 containingthe extracellular domain does not inhibit TER after tight junctiondisruption, contrary to published results for murine JAM-1. On thisbasis it is considered that the data may not support a model ofextracellular homo- or heterotypic interaction mediated by the humanJAM-1 extracellular domain. In another study investigating thestructure/function of human JAM-1, Williams et al. Mol. Immunol.36:1175-1188 (1999) report that both murine and human JM Fc chimeras andtransfected COS cells failed to show homotypic adhesion for the proteinin vitro—suggesting that “firm adhesion may not be the function of thismolecule in vivo.” In a separate study, Liang et al. Am. J. Physiol.279:1733-1743 (2000) report that a recombinant soluble form of humanJAM-1 inhibits recovery of TER following trypsin-EDTA disruption of TJs.The following shows the amino acid sequence of JAM-1 in which theextracellular domain is underlined. Human JAM-1: 299 amino acids.    MGTKAQVERKLLCLFILAILLCSLALG SVTVH (SEQ ID NO: 1)SSEPEVRIPENNPVKLSCAYSGFSSPRVEWKFDQGDTTRLVCYNNKITASYEDRVTFLPTGITFKSVTREDTGTYTCMVSEEGGNSYGEVKVKLIVLVPPSKPTVNIPSSATIGNRAVLTCSEQDGSPPSEYTWFKDGIVMPTNPKSTRAFSNSSYVLNPTTGELVFDPLSASDTGEYSC EARNGYGTPMTSNAVRMEAVERNVGVIVAAVLVTLI LLGILVFGIWFAYSRGHFDRTKKGTSSKKVIYSQPS ARSEGEFKQTSSFLV

The following shows the amino acid sequence of claudin-4 in which theextracellular domain of the protein is underlined. Human Claudin-4: 209amino acids MASMGLQVMGIALAVLGWLAVMLCCALPMW RVTAFI (SEQ ID NO: 2)GSNIVTSQTIWEGLWMNCVVQSTGQMQCKVYDSLLA LPQDLQAARALVIISIIVAALGVLLSVVGGKCTNCL EDESAKAKTMIVAGVVFLLAGLMVIVPVSWTAHNIIQDFYNPLVASGQKRE MGASLYVGWAASGLLLLGGGL LCCNCPPRTDKPYSAKYSAARSAAASNYV

The following shows the amino acid sequence of human occludin in whichthe extracellular domain of the protein is underlined. Human Occludin:522 amino acids. MSSRPLESPPPYRPDEFKPNHYAPSNDIYGGEMH (SEQ ID NO: 56)VRPMLSQPAYSFYPEDEILHFYKWTSPPGVIRIL SMLIIVMCIAIFACVASTLAW DRGYGTSLLGGSVGYPYGGSGFGSYGSGYGYGYGYGYGYGGYTDPR A AKGFMLAMAAFCFIAALVIFVTSVIRSEMSRTRRYYLSVIIVSAILGIMVFIATIVYIM GVNPTAQSS GSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQE AIAIVLGFMIIVAFALIIFFAVKTRRKMD RYDKSNILWDKEHIYDEQPPNVEEWVKNVSAGTQDVPSPPSDYVERVDSPMAYSSNGKVNDKRFYPES SYKSTPVPEVVQELPLTSPVDDFRQPRYSSGGNFETPSKRAPAKGRAGRSKRTEQDHYETDYTTGGES CDELEEDWIREYPPITSDQQRQLYKRNFDTGLQEYKSLQSELDEINKELSRLDKELDDYREESEEYMA AADEYNRLKQVKGSADYKSKKNHCKQLKSKLSHIKKMVGDYDRQKT

Thus, the invention also provides diagnostic and therapeutic antibodies,including monoclonal antibodies, directed against a JAM, occludin orclaudin peptide or protein, including antibodies against specificportions or domains (e.g., a homotypic binding interface) of a JAM,occludin or claudin protein. The antibodies specifically recognizefunctional portions of the JAM, occludin or claudin protein, and aretherefore useful for blocking interactions between these proteins, orpermeabilizing mucosal epithelial target cells when administered invivo. These immunotherapeutic reagents may include humanized antibodies,and can be combined for therapeutic use with additional active or inertingredients as disclosed herein, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, e.g., immunogenic adjuvants, andoptionally with adjunctive or combinatorially active agents such asantiretroviral drugs. Methods for generating functional antibodies,including humanized antibodies, antibody fragments, and other relatedagents are well known in the art (see, e.g., Harlow & Lane, Antibodies,A Laboratory Manual, (CSHP, NY, 1988); Queen et al., Proc. Natl. Acad.Sci. USA 86:10029-10033 (1989) and WO 90/07861. Human antibodies can beobtained using phage-display methods (see, e.g., Dower et al., WO91/17271; McCafferty et al., WO 92/01047). Similarly, methods forproducing active antibody fragments are well known, including methodsfor generating separate heavy chains, light chains Fab, Fab′ F(ab′)2,Fv, and single chain antibodies. Fragments can be produced by enzymaticor chemical separation of intact immunoglobulins using standard methods.Fab fragments may be obtained from F(ab′)2 fragments by limitedreduction, or from whole antibody by digestion with papain in thepresence of reducing agents. Fragments can also be produced byrecombinant DNA techniques. Segments of nucleic acids encoding selectedfragments are produced by digestion of full-length coding sequences withrestriction enzymes, or by de novo synthesis. Often fragments areexpressed in the form of phage-coat fusion proteins. This manner ofexpression is advantageous for affinity-sharpening of antibodies.

According to the present invention, the tight junctions can also beopened using RNAi techniques. RNA interference refers to the process ofsequence-specific post-transcriptional gene silencing in animalsmediated by short interfering nucleic acids (siNAs), which are usuallyshort interfering RNAs (siRNA). See Fire et al., Nature, 391:806 (1998)and Hamilton et al., Science, 286: 950-951 (1999). The correspondingprocess in plants is commonly referred to as post-transcriptional genesilencing or RNA silencing and is also referred to as quelling in fungi.The process of post-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse flora andphyla [Fire et al., Trends Genet., 15: 358 (1999)]. Such protection fromforeign gene expression may have evolved in response to the productionof double-stranded RNAs (dsRNAs) derived from viral infection or fromthe random integration of transposon elements into a host genome via acellular response that specifically destroys homologous single-strandedRNA or viral genomic RNA. The presence of dsRNA in cells triggers theRNAi response though a mechanism that has yet to be fully characterized.This mechanism appears to be different from the interferon response thatresults from dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) [Hamilton et al., supra; Berstein et al.,Nature, 409: 363(2001)]. Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes [Hamilton et al., supra; Elbashiret al., Genes Dev., 15: 188 (2001)]. The siRNA molecules bind to aprotein complex termed RNA-induced silencing complex (RISC), whichcontains a helicase activity that unwinds the two strands of the siRNAmolecules. RISC will then incorporate one strand into its complex, whichstrand will hybridize to a target mRNA and the RISC will hydrolyze orcleave the target mRNA at the site where the antisense strand is bound.Cleavage of the target RNA takes place in the middle of the regioncomplementary to the antisense strand of the siRNA duplex [Elbashir etal., 2001, Genes Dev., 15, 188 (2001)].

Elbashir et al., Nature, 411: 494 (2001), describe RNAi induced byintroduction of duplexes of synthetic 21 -nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork in Drosophila embryonic lysates [Elbashir et al., EMBO J, 26: 6877(2001)] has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21-nucleotidesiRNA duplexes are most active when containing 3′-terminal dinucleotideoverhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAiactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated.Single mismatch sequences in the center of the siRNA duplex were alsoshown to abolish RNAi activity. In addition, these studies also indicatethat the position of the cleavage site in the target RNA is defined bythe 5′-end of the siRNA guide sequence rather than the 3′-end of theguide sequence [Elbashir et al., EMBO J, 20: 6877 (2001)]. Other studieshave indicated that a 5′-phosphate on the target-complementary strand ofa siRNA duplex is required for siRNA activity and that ATP is utilizedto maintain the 5′-phosphate moiety on the siRNA [Nykanen et al., Cell,107: 309 (2001)].

Nucleic acid molecules that act as mediators of the RNA interferencegene silencing response are double-stranded nucleic acid molecules. Thedouble-stranded nucleic acids are generally double-stranded RNA,however, the strands can contain one or more deoxyribonucleotides. Themore precise term would be to call the RNAi mediators, small interferingnucleic acids or ‘siNA’. The siNA molecules are comprised of duplexescontaining about 19 base pairs between oligonucleotides comprising about19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24 or 25) nucleotides.The siNA molecules may also be comprised of duplexes with overhangingends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, forexample, about 21-nucleotide duplexes with about 19 base pairs and3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs.

General Description of RNA Interference

The following is a further description of other general aspects anddefinitions relating to RNA interference

Non-limiting examples of chemical modifications that can be made in ansiNA include without limitation phosphorothioate internucleotidelinkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides,2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides,“acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryland/or inverted deoxy abasic residue incorporation. These chemicalmodifications, when used in various siNA constructs, are shown topreserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds.

A composition comprising a siNA molecule may be in a pharmaceuticallyacceptable carrier or diluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

The siNA molecules described herein, the antisense region of a siNAmolecule of the invention can comprise a phosphorothioateinternucleotide linkage at the 3′-end of said antisense region. In anyof the embodiments of siNA molecules described herein, the antisenseregion can comprise about one to about five phosphorothioateinternucleotide linkages at the 5′-end of said antisense region. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs of a siNA molecule of the invention can compriseribonucleotides or deoxyribonucleotides that are chemically-modified ata nucleic acid sugar, base, or backbone. In any of the embodiments ofsiNA molecules described herein, the 3′-terminal nucleotide overhangscan comprise one or more universal base ribonucleotides. In any of theembodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more acyclic nucleotides.

For example, in a non-limiting example, the invention features achemically-modified short interfering nucleic acid (siNA) having about1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkagesin one siNA strand. In yet another embodiment, the invention features achemically-modified short interfering nucleic acid (siNA) individuallyhaving about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in both siNA strands. The phosphorothioateinternucleotide linkages can be present in one or both oligonucleotidestrands of the siNA duplex, for example in the sense strand, theantisense strand, or both strands. The siNA molecules of the inventioncan comprise one or more phosphorothioate internucleotide linkages atthe 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sensestrand, the antisense strand, or both strands. For example, an exemplarysiNA molecule of the invention can comprise about 1 to about 5 or more(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioateinternucleotide linkages at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

An siNA molecule may be comprised of a circular nucleic acid molecule,wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50,55, 60, 65, or 70) nucleotides in length having about 18 to about 23(e.g., about 18, 19, 20, 21, 22, or 23) base pairs wherein the circularoligonucleotide forms a dumbbell shaped structure having about 19 basepairs and 2 loops.

A circular siNA molecule contains two loop motifs, wherein one or bothloop portions of the siNA molecule is biodegradable. For example, acircular siNA molecule of the invention is designed such thatdegradation of the loop portions of the siNA molecule in vivo cangenerate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

Modified nucleotides present in siNA molecules, preferably in theantisense strand of the siNA molecules, but also optionally in the senseand/or both antisense and sense strands, comprise modified nucleotideshaving properties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides. 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

The sense strand of a double stranded siNA molecule may have a terminalcap moiety such as an inverted deoxyabasic moiety, at the 3′-end,5′-end, or both 3′ and 5′-ends of the sense strand.

Non-limiting examples of conjugates include conjugates and ligandsdescribed in Vargeese et al., U.S. patent application Ser. No.10/427,160, filed Apr. 30, 2003, incorporated by reference herein in itsentirety, including the drawings. In another embodiment, the conjugateis covalently attached to the chemically-modified siNA molecule via abiodegradable linker. In one embodiment, the conjugate molecule isattached at the 3′-end of either the sense strand, the antisense strand,or both strands of the chemically-modified siNA molecule. In anotherembodiment, the conjugate molecule is attached at the 5′-end of eitherthe sense strand, the antisense strand, or both strands of thechemically-modified siNA molecule. In yet another embodiment, theconjugate molecule is attached both the 3′-end and 5′-end of either thesense strand, the antisense strand, or both strands of thechemically-modified siNA molecule, or any combination thereof. In oneembodiment, a conjugate molecule of the invention comprises a moleculethat facilitates delivery of a chemically-modified siNA molecule into abiological system, such as a cell. In another embodiment, the conjugatemolecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellularreceptor that can mediate cellular uptake. Examples of specificconjugate molecules contemplated by the instant invention that can beattached to chemically-modified siNA molecules are described in Vargeeseet al., U.S. patent application Publication No. 20030130186, publishedJul. 10, 2003, and U.S. patent application Publication No. 20040110296,published Jun. 10, 2004. The type of conjugates used and the extent ofconjugation of siNA molecules of the invention can be evaluated forimproved pharmacokinetic profiles, bioavailability, and/or stability ofsiNA constructs while at the same time maintaining the ability of thesiNA to mediate RNAi activity. As such, one skilled in the art canscreen siNA constructs that are modified with various conjugates todetermine whether the siNA conjugate complex possesses improvedproperties while maintaining the ability to mediate RNAi, for example inanimal models as are generally known in the art.

A siNA further may be further comprised of a nucleotide, non-nucleotide,or mixed nucleotide/non-nucleotide linker that joins the sense region ofthe siNA to the antisense region of the siNA. In one embodiment, anucleotide linker can be a linker of>2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule where thetarget molecule does not naturally bind to a nucleic acid. The targetmolecule can be any molecule of interest. For example, the aptamer canbe used to bind to a ligand-binding domain of a protein, therebypreventing interaction of the naturally occurring ligand with theprotein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. [See, for example, Gold et al,Annu. Rev. Biochem., 64: 763 (1995); Brody and Gold, J. Biotechnol., 74:5 (2000); Sun, Curr. Opin. Mol. Ther., 2:100 (2000); Kusser, J.Biotechnol., 74: 27 (2000); Hermann and Patel, Science 287: 820 (2000);and Jayasena, Clinical Chemistry, 45: 1628. (1999)

A non-nucleotide linker may be comprised of an abasic nucleotide,polyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycolssuch as those having between 2 and 100 ethylene glycol units). Specificexamples include those described by Seela and Kaiser, Nucleic AcidsRes., 18:6353 (1990) and Nucleic Acids Res., 15:3113 (1987); Cload andSchepartz, J. Am. Chem. Soc., 113:6324 (1991); Richardson and Schepartz,J. Am. Chem. Soc., 113:5109 (1991); Ma et al., Nucleic Acids Res.,21:2585 (1993) and Biochemistry 32:1751(1993); Durand et al., NucleicAcids Res., 18:6353 (1990); McCurdy et al., Nucleosides & Nucleotides,10:287 (1991); Jschke et al., Tetrahedron Lett., 34:301 (1993); Ono etal., Biochemistry, 30:9914 (1991); Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc., 113:4000 (1991). A“non-nucleotide” further means any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound can be abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine, for example at the C1 position of the sugar.

The synthesis of a siNA molecule of the invention, which can bechemically-modified, comprises: (a) synthesis of two complementarystrands of the siNA molecule; (b) annealing the two complementarystrands together under conditions suitable to obtain a double-strandedsiNA molecule. In another embodiment, synthesis of the two complementarystrands of the siNA molecule is by solid phase oligonucleotidesynthesis. In yet another embodiment, synthesis of the two complementarystrands of the siNA molecule is by solid phase tandem oligonucleotidesynthesis.

Synthesis of Nucleic Acid Molecules

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. RNA including certain siNA moleculesof the invention follows the procedure as described in Usman et al.,1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic AcidsRes., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23,2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59.

Administration of Nucleic Acid Molecules

Methods for the delivery of nucleic acid molecules are described inAkhtar et al., Trends Cell Bio., 2, 139 (1992); Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,Mol. Membr. Biol., 16: 129-140 (1999); Hofland and Huang, Handb. Exp.Pharmacol., 137: 165-192 (1999); and Lee et al., ACS Symp. Ser., 752:184-192 (2000), Sullivan et al., PCT WO 94/02595, further describes thegeneral methods for delivery of enzymatic nucleic acid molecules. Theseprotocols can be utilized for the delivery of virtually any nucleic acidmolecule. Nucleic acid molecules can be administered to cells by avariety of methods known to those of skill in the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (O'Hare and Normand, International PCT PublicationNo. WO 00/53722). Alternatively, the nucleic acid/vehicle combination islocally delivered by direct injection or by use of an infusion pump.Direct injection of the nucleic acid molecules of the invention, whethersubcutaneous, intramuscular, or intradermal, can take place usingstandard needle and syringe methodologies, or by needle-freetechnologies such as those described in Conry et al., Clin. Cancer Res.,5: 2330-2337 (1999) and Barry et al., International PCT Publication No.WO 99/31262. The molecules of the instant invention can be used aspharmaceutical agents. Pharmaceutical agents prevent, modulate theoccurrence, or treat (alleviate a symptom to some extent, preferably allof the symptoms) of a disease state in a patient.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The negatively chargedpolynucleotides of the invention can be administered (e.g., RNA, DNA orprotein) and introduced into a patient by any standard means, with orwithout stabilizers, buffers, and the like, to form a pharmaceuticalcomposition. When it is desired to use a liposome delivery mechanism,standard protocols for formation of liposomes can be followed. Thecompositions of the present invention may also be formulated and used astablets, capsules or elixirs for oral administration, suppositories forrectal administration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or patient, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes which lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes expose the desired negativelycharged polymers, e.g., nucleic acids, to an accessible diseased tissue.The rate of entry of a drug into the circulation has been shown to be afunction of molecular weight or size. The use of a liposome or otherdrug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach may provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Nonlimiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS [Jolliet-Riant andTillement, Fundam. Clin. Pharmacol., 13:16-26 (1999)]; biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich, DF et al., Cell Transplant, 8: 47-58 (1999)] (Alkermes, Inc. Cambridge,Mass.); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23: 941-949, (1999)]. Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., J. Pharm. Sci., 87:1308-1315 (1998); Tyler et al., FEBS Lett.,421: 280-284 (1999); Pardridge et al., PNAS USA., 92: 5592-5596 (1995);Boado, Adv. Drug Delivery Rev., 15: 73-107 (1995); Aldrian-Herrada etal., Nucleic Acids Res., 26: 4910-4916 (1998); and Tyler et al., PNASUSA., 96: 7053-7058 (1999).

The present invention also includes compositions prepared for storage oradministration, which include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).For example, preservatives, stabilizers, dyes and flavoring agents maybe provided. These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentsmay be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence of, or treat (alleviate a symptom to some extent,preferably all of the symptoms) a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

The pharmaceutical compositions can be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension can beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents that have been mentioned above. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a non-toxic parentally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil can be employed including synthetic mono-ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

The siNAs can also be administered in the form of suppositories, e.g.,for rectal administration of the drug. These compositions can beprepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and polyethylene glycols.

The siNAs can be modified extensively to enhance stability bymodification with nuclease resistant groups, for example, 2′-amino,2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H. [For a review see Usman andCedergren, TIBS 17: 34 (1992); Usman et al., Nucleic Acids Symp. Ser.31: 163 (1994)]. SiNA constructs can be purified by gel electrophoresisusing general methods or can be purified by high pressure liquidchromatography and re-suspended in water.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency. See e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., Nature344: 565 (1990); Pieken et al., Science 253, 314 (1991); Usman andCedergren, Trends in Biochem. Sci. 17: 334 (1992); Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074. All of the abovereferences describe various chemical modifications that can be made tothe base, phosphate and/or sugar moieties of the nucleic acid moleculesdescribed herein.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications. For areview see Usman and Cedergren,, TIBS. 17: 34 (1992); Usman et al.,Nucleic Acids Symp. Ser. 31:163 (1994); Burgin et al., Biochemistry, 35:14090 (1996). Sugar modification of nucleic acid molecules have beenextensively described in the art. See Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature,344, 565-568(1990); Pieken et al. Science, 253: 314-317 (1991); Usman and Cedergren,Trends in Biochem. Sci., 17: 334-339 (1992); Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al.,Karpeisky et al., Tetrahedron Lett., 39: 1131 (1998); Earnshaw and Gait,Biopolymers (Nucleic Acid Sciences), 48: 39-55 (1998); Verma andEckstein, Annu. Rev. Biochem., 67: 99-134 (1998); and Burlina et al.,Bioorg. Med. Chem., 5: 1999-2010 (1997). Such publications describegeneral methods and strategies to determine the location ofincorporation of sugar, base and/or phosphate modifications and the likeinto nucleic acid molecules without modulating catalysis. In view ofsuch teachings, similar modifications can be used as described herein tomodify the siNA nucleic acid molecules of the instant invention so longas the ability of siNA to promote RNAi in cells is not significantlyinhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417 (1995), and Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994).

Methods for the delivery of nucleic acid molecules are described inAkhtar et al., Trends Cell Bio., 2: 139 (1992); Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed. Akhtar, (1995), Maurer etal., Mol. Membr. Biol., 16: 129-140 (1999); Hofland and Huang, Handb.Exp. Pharmacol., 137: 165-192 (1999); and Lee et al., ACS Symp. Ser.,752: 184-192 (2000). Beigelman et al., U.S. Pat. No. 6,395,713 andSullivan et al., PCT WO 94/02595 further describe the general methodsfor delivery of nucleic acid molecules. These protocols can be utilizedfor the delivery of virtually any nucleic acid molecule. Nucleic acidmolecules can be administered to cells by a variety of methods known tothose of skill in the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as biodegradable polymers, hydrogels, cyclodextrins(see for example Gonzalez et al., Bioconjugate Chem., 10: 1068-1074(1999); Wang et al., International PCT publication Nos. WO 03/47518 andWO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres(see for example U.S. Pat. No. 6,447,796 and U.S. patent applicationPublication No. US 2002130430), biodegradable nanocapsules, andbioadhesive microspheres, or by proteinaceous vectors (O'Hare andNormand, International PCT Publication No. WO 00/53722). Alternatively,the nucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of the invention, whether subcutaneous, intramuscular, orintradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies such as those described inConry et al., Clin. Cancer Res., 5: 2330-2337 (1999) and Barry et al.,International PCT Publication No. WO 99/31262. The molecules of theinstant invention can be used as pharmaceutical agents. Pharmaceuticalagents prevent, modulate the occurrence, or treat (alleviate a symptomto some extent, preferably all of the symptoms) of a disease state in asubject.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner For example the siNA canbe a double-stranded polynucleotide molecule comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof. The siNA can be assembledfrom two separate oligonucleotides, where one strand is the sense strandand the other is the antisense strand, wherein the antisense and sensestrands are self-complementary (i.e. each strand comprises nucleotidesequence that is complementary to nucleotide sequence in the otherstrand; such as where the antisense strand and sense strand form aduplex or double stranded structure, for example wherein the doublestranded region is about 19 base pairs); the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. Alternatively, the siNA is assembled froma single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s). The siNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., Cell., 110-563-574 (2002) and Schwarz et al.,Molecular Cell, 10: 537-568(2002), or 5′,3′-diphosphate. In certainembodiment, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. In certain embodiments short interfering nucleic acids donot require the presence of nucleotides having a 2′-hydroxy group formediating RNAi and as such, short interfering nucleic acid molecules ofthe invention optionally do not include any ribonucleotides (e.g.,nucleotides having a 2′-OH group). Such siNA molecules that do notrequire the presence of ribonucleotides within the siNA molecule tosupport RNAi can however have an attached linker or linkers or otherattached or associated groups, moieties, or chains containing one ormore nucleotides with 2′-OH groups. Optionally, siNA molecules cancomprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of thenucleotide positions. As used herein, the term siNA is meant to beequivalent to other terms used to describe nucleic acid molecules thatare capable of mediating sequence specific RNAi, for example shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA),short hairpin RNA (shRNA), short interfering oligonucleotide, shortinterfering nucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure to alter gene expression. See, for example,Allshire, Science, 297,: 1818-1819(2002); Volpe et al., Science, 297:1833-1837 (2002); Jenuwein, Science, 297: 2215-2218 (2002); and Hall etal., Science, 297: 2232-2237 (2002).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a T-cell (e.g. about 19 toabout 22 (e.g., about 19, 20, 21, or 22) nucleotides) and a loop regioncomprising about 4 to about 8 (e.g., about 4, 5, 6, 7, or 8)nucleotides, and a sense region having about 3 to about 18 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotidesthat are complementary to the antisense region. The asymmetric hairpinsiNA molecule can also comprise a 5′-terminal phosphate group that canbe chemically modified. The loop portion of the asymmetric hairpin siNAmolecule can comprise nucleotides, non-nucleotides, linker molecules, orconjugate molecules as described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina T-cell (e.g. about 19 to about 22 (e.g. about 19, 20, 21, or 22)nucleotides) and a sense region having about 3 to about 18 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotidesthat are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence.

By “highly conserved sequence region” is meant, a nucleotide sequence ofone or more regions in a target gene does not vary significantly fromone generation to the other or from one biological system to the other.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art [see, e.g., Turner et al., CSH Symp.Quant. Biol. LII pp.123-133 (1987); Frier et al., Proc. Nat. Acad. Sci.USA 83:9373-9377 (1986); Turner et al., J. Am. Chem. Soc. 109:3783-3785(1987)]. A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire-multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing.- The cell can also be derived from or can comprise agamete or embryo, a stem cell, or a fully differentiated cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising.” Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of.”Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By“consisting essentially of” is meant including any elements listed afterthe phrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a .beta.-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art [see for example Loakes, Nucleic Acids Research, 29,2437-2447 (2001)].

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203). These terminalmodifications protect the nucleic acid molecule from exonucleasedegradation, and may help in delivery and/or localization within a cell.The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal(3′-cap) or may be present on both termini. In non-limiting examples,the 5′-cap includes, but is not limited to, glyceryl, inverted deoxyabasic residue (moiety); 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Lyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al, International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra). There are several examples of modified nucleicacid bases known in the art as summarized by Limbach et al, NucleicAcids Res. 22, 2183 (1994). Some of the non-limiting examples of basemodifications that can be introduced into nucleic acid moleculesinclude, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, and others (Burgin et al., Biochemistry, 35:14090 (1996); Uhlman & Peyman, supra). By “modified bases” in thisaspect is meant nucleotide bases other than adenine, guanine, cytosineand uracil at 1′ position or their equivalents.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by a siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human,animal, plant, insect, bacterial, viral or other sources, wherein thesystem comprises the components required for RNAi activity. The term“biological system” includes, for example, a cell, tissue, or organism,or extract thereof. The term biological system also includesreconstituted RNAi systems that can be used in an in vitro setting.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thesense and antisense strands of a siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon of.beta.-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878.

The siNA molecules can be complexed with cationic lipids, packagedwithin liposomes, or otherwise delivered to target cells or tissues. Thenucleic acid or nucleic acid complexes can be locally administered tothrough injection, infusion pump or stent, with or without theirincorporation in biopolymers. In another embodiment, polyethylene glycol(PEG) can be covalently attached to siNA compounds of the presentinvention. The attached PEG can be any molecular weight, preferably fromabout 2,000 to about 50,000 Daltons (Da).

The sense region can be connected to the antisense region via a linkermolecule, such as a polynucleotide linker or a non-nucleotide linker.

EXAMPLE 1

Tight junctions (TJ) form at the apical end of lateral membranes asclosed contacts between the plasma membranes of neighboring cells. TheTJ serves as a diffusion barrier between compartments and it is crucialfor the development and function of epithelial tissues. Previous studieshave suggested that the intra-membrane connections are formed bynon-covalently linked branched polymers containing Claudins (CLDNs),Occludin and Junctional Adhesion Molecules (JAMs). These transmembraneproteins interact with multiple components of a cytoplasmic plaqueconsisting of different types of cytosolic proteins that interact withan actin-based cytoskeleton. There are at least 20 different claudins inepithelial tissues. CLDN tissue-specific expression has beendemonstrated in normal as well as in malignant epithelial tissues.

Studies of the structure and functional properties of the TJ will notonly provide insight on how the barrier is formed, but will identifythose components that are most amenable to functional manipulation. Theability to transiently open tight junctions will facilitate the deliveryof large molecule drugs, such as peptides, for nasal drug delivery.

Experimental Methods

Cell Culture—16HBE14o- Cells were grown in collagen coated flasks incomplete MEM supplemented with 10% FBS. During passage, cells werewashed with PBS and detached using a low concentration of trypsinsolution. For Insert seeding, cells were re-suspended in DMEM/F12complete media. 12-well BD Falcon inserts (Becton Dickinson, FranklinLakes, N.J.) with 0.4 μm pore size were presoaked with media. Cellsuspension was then added onto the apical side and cultured for 72 hrsbefore transfection. TER changes were monitored to evaluate the TJformation. siRNA Transfection—Four hour transfections of siRNAs wereconducted 72 and 96 hours after seeding cells on inserts in serum freeDMEM/F12, followed by addition of complete media. Each transfectionmixture contained 80 pmol of siRNA and 4 μl of Lipofectamine 2000(Invitrogen, Carlsbad, Calif.). Cells grown in dishes were transfectedat approximately 50% confluence with 100 pmol siRNA /well in 6-wellplates. Cells were then allowed to grow to confluence after transfectionand either harvested for insert seeding or photographed. For insertseeding with siRNA-treated cells, transfected cells were detached,counted and re-suspended prior to seeding. When over 80 pmol siRNA wasused for each insert in optimal lipofectamine/siRNA ratio fortransfection, cell damage was observed as indicated by MTT and LDHassays. TER Measurement—Fresh, warm media is placed in a UV sterilizedEndohm tissue resistance measurement chamber connected to an EVOMresistance meter from WPI (Sarasota, Fla.), and allowed to equilibrateto room temperature. Inserts to be measured were removed from theincubator and allowed to equilibrate to room temperature prior tomeasurement.

Permeability Assay—Inserts were left in the TER chamber until constantvalues were displayed. 50 μg of FITC dextran, MW 4,400, was applied onthe apical side of each insert. Medium from basolateral chamber wastransferred into 96-well plates and the amount of FITC determined bymeasuring fluorescence intensity following 1 hr incubation Thepermeability of each sample was calculated as percent of control (totalfluorescence from lysed cell insert).

Immunofluorescence—Cells were either grown on collagen V coatedcoverslips or multi-well plates. After transfection or other treatments,cells were rinsed with PBS twice and fixed with 1% formaldehyde in PBSat room temperature for 10 min. Then 0.2% Triton X-100 was used topermeabilize cells for 10 min at room temperature. The cells were thenblocked with 1% BSA for 30 min at room temperature and incubated withprimary antibodies against Occludin or Claudin 4 (Zymed Inc. South SanFrancisco, Calif.) at room temperature for 60 min. FITC-conjugated goatanti-rabbit IgG (1:200) was used for Occludin and TRITC-conjugated goatanti-mouse (1:200) for Claudin 4 in double labeling at room temperaturefor 30 min. Between steps, the cells were washed 3×-PBS. Vectashieldmounting medium with DAPI (Vector Laboratories, Burlingame, Calif.) wereused for coverslips. The images were captured using an invertedfluorescence microscope (Nikon eclipse TE2000). TABLE 1 ExpressionProfile of Claudins in Differentiated and Undifferentiated EpithelialCells Undiffrentiated Differentiated RPMI 16HBE14 Claudins Product TBE2650 AIR o- CLDN-1 208 ++++ ++++ ++++ ++++ CLDN-2 203 + + − + CLDN-3 247− − +++++ ++++ CLDN-4 217 + − ++++ ++++ CLDN-5 221 + − + ++ CLDN-6 150 −− − − CLDN-7 196 ++ ++ ++++ ++++ CLDN-8 204 − − − − CLDN-9 225 − − ++++− CLDN-10 238 − − + + CLDN-11 235 ++ − ++ + CLDN-12 150 ++++ ++++ ++++++++ CLDN-14 235 + + ++ + CLDN-15 186 − − − − CLDN-16 204 − ++ + +CLDN-17 246 − − − − CLDN-18 190 − + − − CLDN-19 204 − − − − CLDN-20 156++++ ++ ++++ ++Scale of expression level (+) is relative to actin.TBE- undifferentiated cells from MatTek;Air- differentiated cells from MatTek

TABLE 2 siRNA Sequences for Tight Junction Proteins TJ Proteins siRNATarget Sequence Claudin 1 AACCUCUUACCCAACACCAAG (SEQ ID NO: 3) Claudin 3AAAUCACGUCGCAGAACAUUU (SEQ ID NO: 4) Claudin 4 AAGACUUCUACAAUCCGCUGG(SEQ ID NO; 5) Claudin 9 AAGGUGUACGACUCACUGCUG (SEQ ID NO: 6) Claudin 12AAUAGUGCAGGUUGCCACCUG (SEQ ID NO: 7) Claudin 20 AAUGAAAUGUACUCGCUUAGG(SEQ ID NO: 8) JAM-1 GACCUUCUUGCCAACUGGUAU (SEQ ID NO; 9) OccludinGAAAACUCGAAGAAAGAUGGA (SEQ ID NO: 10) Negative AAUUCUCCGAACGUGUCACGU(SEQ ID NO; 11) Control

The siRNAs used to target the mRNA of the tight junction proteins werethe following. siRNA sequences Targets RNA sense sequence Antisensesequence Claudin-1 CCUCUUACCCAACACCAAG GAACCACAACCCAUUCUCC (SEQ ID NO:12) (SEQ ID NO: 13) Claudin-1 GCAUGGUAUGGCAAUAGAA UUCUAUUGCCAUACCAUGC(SEQ ID NO: 14) (SEQ ID NO: 15) Claudin-3 CAUCAUCACGUCGCAGAACGUUCUGCGACGUGAUGAUG (SEQ ID NO: 16) (SEQ ID NO: 17) Claudin-3GCAAGGACUACCGUCUAUU AAUAGACGGUAGUCCUUGC (SEQ ID NO: 18) (SEQ ID NO: 19)Claudin-3 AUCACGUCGCAGAACAUUU AAAUGUUCUGCGACGUGAU (SEQ ID NO: 20) (SEQID NO: 21) Claudin-4 GACUUCUACAAUCCGCUGG CCAGCGGAUUGUAGAAGUC (SEQ ID NO:22) (SEQ ID NO: 23) Claudin-4 CAUCAUCCAAGACUUCUAC GUAGAAGUCUUGGAUGAUG(SEQ ID NO: 24) (SEQ ID NO: 25) Claudin-4 GACUUCUACAAUCCGCUGGCCAGCGGAUUGUAGAAGUC (SEQ ID NO: 26) (SEQ ID NO: 27) Claudin-9CCCACUUUCCAAAAGCCCA UGGGCUUUUGGAAAGUGGG (SEQ ID NO: 28) (SEQ ID NO: 29)Claudin-9 GGUGUACGACUCACUGCUG CAGCAGUGAGUCGUACACC (SEQ ID NO: 30) (SEQID NO: 31) Claudin-12 UAGUGCAGGUUGCCACCUG CAGGUGGCAACCUGCACUA (SEQ IDNO: 32) (SEQ ID NO: 33) Claudin-12 GUGACUGCCUGAUGUACGAUCGUACAUCAGGCAGUCAC (SEQ ID NO: 34) (SEQ ID NO: 35) Claudin-12AUGCGCAACACUGCCUUCA UGAAGGCAGUGUUGCGCAU (SEQ ID NO: 36) (SEQ ID NO: 37)Claudin-20 AUGUACUCGCUUAGGAGGG CCCUCCUAAGCGAGUACAU (SEQ ID NO: 38) (SEQID NO: 39) Claudin-20 UGAAAUGUACUCGCUUAGG CCUAAGCGAGUACAUUUCA (SEQ IDNO: 40) (SEQ ID NO: 41) Claudin-20 CCUGGAGGAGCUAUCUAUAUAUAGAUAGCUCCUCCAGG (SEQ ID NO: 42) (SEQ ID NO: 43) OccludinUGGGAGUGAACCCAACUGC GCAGUUGGGUUCACUCCCA (SEQ ID NO: 44) (SEQ ID NO: 45)Occludin AAACUCGAAGAAAGAUGGA UCCAUCUUUCUUCGAGUUU (SEQ ID NO: 46) (SEQ IDNO: 47) Occludin ACAGAGCAAGAUCACUAUG CAUAGUGAUCUUGCUCUGU (SEQ ID NO: 48)(SEQ ID NO: 49) JAM-1 UCCCACAACAGGAGAGCUG CAGCUCUCCUGUUGUGGGA (SEQ IDNO: 50) (SEQ ID NO: 51) JAM-1 CCUUCUUGCCAACUGGUAU AUACCAGUUGGCAAGAAGG(SEQ ID NO: 52) (SEQ ID NO: 53) JAM-1 GCCUCUGAUACUGGAGAAUAUUCUCCAGUAUCAGAGGC (SEQ ID NO: 54) (SEQ ID NO: 55)The dTdT are added to 3′ ends of both sense and antisense strands

SUMMARY

-   -   Claudins 3 and 4 are differentially expressed in differentiated        primary (MatTeck) and immortalized tight junction-forming cells        (16HBE14o-), while Claudins 1, 12 and 20 are expressed in both        tight junction forming cells and non-tight junction forming        cells (BET cells from MatTeck and RPM12650, a nasal tumor        epithelial cells).    -   At least one effective siRNA was identified for Claudins 1, 3,        4, 9, 12, 20, JAM-1, and Occludin in respiratory epithelial        cells.    -   Claudin 4 was found to be essential to maintain tight junction        integrity. Knocking down Claudin 4 expression either before or        after tight junction formation resulted in a significant        decrease in TER and this effect correlated with increased        permeability for dextran, MW 4,400. Lesser effects on TER were        observed for Occludin and JAM 1, while knockdown of Claudin 12        had little effect on TER.    -   Knockdown of Claudin 4 affected cell phenotype (FIG. 9) as        evidenced by changes in cell shape and a disorganized growth        pattern. siRNA-transfected 16HBE14o-cells exhibited multilayer        growth compared to untransfected cells which grew as a        monolayer.    -   When cells were transfected with siRNA against Claudin 4 in        combination with either occludin or JAM-1 (40 pmol of siRNAs),        the effects on TER appeared to be synergistic. In contrast,        combinations with siRNA against Claudin 12 showed little effect.

The teachings of all of the references cited herein are incorporated intheir entirety by reference.

1. A formulation for administering a drug intranasally comprised of adrug, an antagonist that binds to an extracellular domain of a JAM-1protein or an antagonist that binds to an extracellular domain of aclaudin-4 protein or an antagonist that binds to an extracellular domainof a occludin protein.
 2. The formulation of claim 1 wherein theformulation contains an antagonist of the JAM-1 protein and anantagonist to the claudin-4 protein.
 3. The formulation of claim 1wherein the formulation contains an antagonist of the JAM-1 protein andan antagonist to the occludin protein.
 4. The formulation of claim 1wherein the formulation contains an antagonist of the occludin proteinand an antagonist to the claudin-4 protein.
 5. The formulation of claim1 wherein the formulation contains an antagonist of the JAM-1 protein,and antagonist to the occludin protein and an antagonist to theclaudin-4 protein.
 6. The formulation claim 1 wherein the antagonistsare antibodies, fragments of antibodies or single-chain antibodies.
 7. Amethod for opening tight junctions in a tissue comprising bringing thetissue into with an antagonist that binds to an extracellular domain ofa JAM-1 protein or an antagonist that binds to an extracellular domainof a claudin-4 protein or an antagonist that binds to an extracellulardomain of a occludin protein.
 8. The method of claim 7 furthercomprising bringing an antagonist of the JAM-1 protein and an antagonistto the claudin-4 protein into contact with the tissue so as to open thetight junction.
 9. The method of claim 7 further comprising bringing anantagonist of the JAM-1 protein and an antagonist to the occludinprotein into contact with the tissue so as to open the tight junction.10. The method of claim 7 further comprising bringing an antagonist ofthe occludin and an antagonist to the claudin-4 protein into contactwith the tissue so as to open the tight junction.
 11. The method ofclaim 7 further comprising bringing an antagonist of a the JAM-1 proteinand, an antagonist to the occludin protein an antagonist to theclaudin-4 protein into contact with the tissue so as to open the tightjunction.
 12. The method of claim 7 wherein the antagonists areantibodies, fragments of antibodies or single-chain antibodies.
 13. Themethod of claim 7 wherein the tissue is nasal mucosa.
 14. A formulationfor administering a drug intranasally comprised of a drug, a smallinterfering nucleic acid (siNA) that down-regulates expression of aJAM-1 protein or a small interfering nucleic acid that down-regulatesexpression of a claudin-4 protein or a small interfering nucleic acidthat down-regulates expression of a occludin protein.
 15. Theformulation of claim 14 wherein the formulation contains an siNA thatcan down-regulate the expression of the JAM-1 protein and an siNA thatcan down-regulate the expression of the claudin-4 protein.
 16. Theformulation of claim 14 wherein the formulation contains an siNA thatcan down-regulate the expression the JAM-1 protein and an siNA that candown-regulate the expression of the occludin protein.
 17. Theformulation of claim 14 wherein the formulation contains an siNA thatcan down-regulate the expression of the occludin protein and an siNAthat can down-regulate the expression of the claudin-4 protein.
 18. Theformulation of claim 14 wherein the formulation contains an siNA thatcan down-regulate the expression the JAM-1 protein, an siNA that candown-regulate the expression of the claudin-4 protein and an siNA thatcan down-regulate the expression of the occludin protein.
 19. A methodfor opening tight junctions in a tissue comprising bringing the tissueinto with a small interfering nucleic acid (siNA) that down-regulatesexpression of a JAM-1 protein or a small interfering nucleic acid thatdown-regulates expression of a claudin-4 protein or a small interferingnucleic acid that down-regulates expression of a occludin protein. 20.The method of claim 19 further comprising bringing an siNA that candown-regulate the expression of the JAM-1 protein and an siNA that candown-regulate the expression of the claudin-4 protein into contact withthe tissue.
 21. The method of claim 19 further comprising bringing ansiNA that can down-regulate the expression the JAM-1 protein and an siNAthat can down-regulate the expression of the occludin protein intocontact with the tissue.
 22. The method of claim 19 further comprisingbringing an siNA that can down-regulate the expression of the occludinprotein and an siNA that can down-regulate the expression of theclaudin-4 protein into contact with the tissue.
 23. The method of claim19 further comprising bringing an siNA that can down-regulate theexpression of the JAM-1 protein, an siNA that can down-regulate theexpression of the claudin-4 protein and an siNA that can down-regulatethe expression of the occludin protein into contact with the tissue soas to open the tight junction.
 24. The method of claim 19 wherein thetissue is nasal mucosa.